盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定(英文)

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70)。为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C. A. Mey. )O. E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70。实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导。Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性。     

热应激和吡虫啉对大豆蚜hsp70和hsc70基因mRNA表达的影响

【目的】昆虫在高温或农药的胁迫下,通过高效表达热休克蛋白(HSP)等建立应激自我保护机制。本研究为从转录组水平上认识大豆蚜Aphis glycines在热应激和吡虫啉胁迫下hsp70和hsc70 mRNA表达分子机制,进而寻找自我保护应激反应中的薄弱环节,为大豆蚜的生物防治提供理论基础。【方法】采用同源克隆、RACE技术和实时荧光定量PCR等方法研究不同热激时间和热激后不同恢复时间及不同吡虫啉浓度对大豆蚜4龄若虫hsp70和hsc70的表达影响。【结果】37℃热激后,大豆蚜4龄若虫中hsp70表达量先上调,1 h时升至对照组的10.36倍（P<0.05）,然后逐渐下降。同样热激后恢复时间的长短对大豆蚜若蚜中hsp70的表达具有显著影响。热激处理后,大豆蚜若蚜中hsp70立即大量表达,表达量为对照组的8.78倍（P<0.05）,随后表达量下降至对照组水平,而hsc70的表达量并没有显著变化（P>0.05）。大豆蚜若蚜受吡虫啉的胁迫时,其hsp70和hsc70的表达量受吡虫啉的浓度及胁迫的时间的影响,呈现先升高后下降的趋势,具有明显的短期效应。【结论】吡虫啉诱导大豆蚜hsp70和hsc70表达量的上调;而热胁迫对hsp70和hsc70 mRNA具有不同的表达模式,高温可以诱导hsp70的表达,但对hsc70没有明显的诱导作用。     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

热胁迫下八字地老虎hsc70基因在不同组织转录表达差异性分析

【目的】为探讨八字地老虎Xestia c-nigrum(Linnaeus)Xe-hsc70基因表达与高温耐受性之间的关系,比较热胁迫下Xe-hsc70基因在不同组织中诱导表达的差异。【方法】本研究以八字地老虎4龄幼虫为研究对象,采用RT-PCR和RACE技术克隆获得编码热激同源蛋白70(70 ku heat shock cognate,HSC70)的基因(命名为Xe-hsc70)cDNA全序列与基因组DNA序列(Genomic DNA,gDNA),并利用实时荧光定量PCR和Western blotting技术,比较分析不同热胁迫温度及不同热诱导时间下八字地老虎4龄幼虫体内马氏管、中肠、体壁、脂肪体与唾腺5个组织中Xe-hsc70基因在mRNA转录与蛋白表达两水平上相对表达量的变化。【结果】比较分析克隆得到的Xe-hsc70基因cDNA全序列和gDNA序列,结果表明Xe-hsc70基因含有8个内含子,最大内含子(561 bp)位于5′端非编码区,并含有一个类似热激应答原件HSE的核心结构序列(gaatatgCaGAAtgTTCcaGaa),其余内含子(长度在86~218 bp之间不等)均在编码区内,本研究首次报道了八字地老虎hsc70内含子的具体数目及位置。组织差异性分析显示:在常温25℃条件下,Xe-HSC70在脂肪体中表达量最高,在唾腺中表达量最低;经热激诱导后中肠、唾腺与体壁组织中Xe-HSC70的表达量与对照(25℃)相比显著上调,随着热激时间的延长,表达量呈现出先升高后恢复至对照水平的变化趋势,脂肪体和马氏管Xe-HSC70表达量与对照相比无明显变化。【结论】八字地老虎不同组织在应对热胁迫的过程中,抗逆机制具有明显的差异。Xe-HSC70的不断积累是八字地老虎对热胁迫不断适应的一个过程,其组织中Xe-hsc70基因的高表达在八字地老虎抗热胁迫的过程中起着重要作用,并为从分子水平上研究八字地老虎的抗逆机理提供依据。     

家蚕hsp70启动子的克隆及功能研究

该研究通过PCR的方法克隆得到家蚕热激蛋白70基因(Bombyx mori hsp70)的5'侧翼的两个长度分别为538 bp和305 bp的序列hsp70-538和hsp70-305。生物信息分析结果表明这两段序列在TATA序列的上游存在保守的热激元件HSE(heat shock element)CTnGAAnnTTCnAG。采用双荧光报告基因技术研究表明这两段序列在BmN细胞中都表现出热激活性,转基因家蚕实验证明hsp70-305在家蚕个体中也具有热激活性,可以认为这两个片段具有hsp70热激启动子特性。     

复苏植物旋蒴苣苔J结构域蛋白编码基因BhDNAJC2的 克隆、表达与功能

热激蛋白(HSP)是一类在受到逆境刺激后大量表达的蛋白质, 能够帮助蛋白质正确折叠, 促使变性蛋白质降解, 缓解逆境胁迫对生物体的损伤。为揭示热激蛋白在耐旱的复苏植物中的保护作用, 该研究对复苏植物旋蒴苣苔(Boea hygrometrica)HSP40家族中J结构域蛋白BhDNAJC2的编码基因进行了克隆、表达与功能分析。Real-time PCR检测表明, 该基因受脱水、低温、热激等多种逆境条件和脱落酸(ABA)诱导表达。BhDNAJC2-YFP定位于细胞质、内质网和细胞核。过表达BhDNAJC2的拟南芥(Arabidopsis thaliana)株系在干旱、热激、盐胁迫和碱胁迫下均表现出明显的抗逆性。综上所述, BhDNAJC2可能在旋蒴苣苔抗旱、耐热及耐盐碱等胁迫反应中起关键作用。     

植物热激蛋白70

植物热激蛋白70(HsP70)由多基因家族编码.除热胁迫外,其它环境因素如低温、干旱等也能诱导HSP70基因的大量表达.HSP70主要参与新生肽的成熟与分拣、变性蛋白的复性或降解等细胞活动.该文介绍HSP70的结构、功能和调控的研究现状.     

人热休克蛋白70的原核高效表达

将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH50α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及Western blot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X／hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。     

坛紫菜hsp70基因真核表达载体的构建与转化

坛紫菜是极具经济效益的大型海藻,生活在环境多变的潮间带,易受到温度、渗遗压和辐射等因素剧烈变化的影响,经常处于逆境胁迫中.热休克蛋白70(HSP70)是生物体内一种重要且高度保守的应激因子,作为分子伴侣在胁迫条件下首先被诱导出来,在逆境胁迫调节中起着重要的作用.构建紫菜hsp70基因真核表达体系对于了解该基因在紫菜抗逆过程中的作用有重要意义.利用PCR技术扩增得到大小为1.89 kb的坛紫菜hsp70基因,将其克隆至pMD18-T载体上测序.从测序正确的菌株中提取质粒,经限制性内切酶Sma Ⅰ和Not Ⅰ进行消化,将目的基因与真核表达质粒p181AINE连接,获得重组真核表达质粒p181-hsp70.通过菌落PCR、双酶切及测序等方法进行鉴定,结果表明重组真核表达质粒p181-hsp70构建成功.制备酵母感受态,将重组质粒电激转入酿酒酵母中,酵母菌落PCR结果显示电激转入成功.     

拟南芥热激转录因子AtHsfA2调节胁迫反应基因的表达并提高热和氧化胁迫耐性

越来越多的证据表明热胁迫和氧化胁迫间存在着内在联系. 最近的研究发现, 热激转录因子(Hsfs)在联系热和氧化胁迫信号反应中具有重要的作用. 对拟南芥热激转录因子AtHsfA2在热激和氧化胁迫反应的功能进行了分析和鉴定. 利用Northern blot和定量RT-PCR的方法, 我们发现AtHsfA2的表达不仅被热激诱导, 同样也受到氧化胁迫诱导. 对AtHsfA2敲除突变体和超表达植株进行功能分析表明, 突变体降低了基础性和获得性耐热能力以及氧化胁迫耐性, 而超表达植株却增强这些耐性, 并且这些表型的改变与APX1和一些热激蛋白基因的表达, 离子渗漏水平, H2O2的水平和膜过氧化伤害程度的变化相关联. 以上结果表明, 拟南芥热激转录因子AtHsfA2通过调节胁迫反应基因的表达而提高热和氧化胁迫耐性. 因此, 我们提出热激转录因子AtHsfA2在联系热和氧化胁迫信号反应中具有重要的作用.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70

Heat shock proteins of the hsp/hsc70 family are essential chaperones, implicated in the stress response, aging, and a growing number of human diseases. At the molecular level, hsc70s are required for the proper folding and intracellular targeting of polypeptides as well as the regulation of apoptosis. Cytoplasmic members of the hsp/hsc70 family are believed to shuttle between nuclei and cytoplasm; they are found in both compartments of unstressed cells. Our experiments demonstrate that actin filament-destabilizing drugs trigger the nuclear accumulation of hsc70s in unstressed and heat-shocked cells recovering from stress. Using human-mouse heterokaryons, we show that stress inhibits shuttling and sequesters the chaperone in nuclei. The inhibition of hsc70 shuttling upon heat shock is only transient, and transport is reestablished when cells recover from stress. Hsc70 shuttling is controlled by hsc70 retention in the nucleus, a process that is mediated by two distinct mechanisms, ATP-sensitive binding of hsc70s to chaperone substrates and, furthermore, the association with nucleoli. The nucleolar protein fibrillarin and ribosomal protein rpS6 were identified as components that show an increased association with hsc70s in the nucleus upon stress exposure. Together, our data suggest that stress abolishes the exit of hsc70s from the nucleus to the cytoplasm, thereby limiting their function to the nuclear compartment. We propose that during recovery from stress hsc70s are released from nuclear and nucleolar anchors, which is a prerequisite to restore shuttling. nuclear transport; chaperone; nuclear retention; nucleoli     

Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.